Analysis of Beta-Blockers Using UPLC with Accurate-Mass Screening

Importance of the Topic

Beta-adrenergic blocking agents are widely prescribed for cardiovascular disorders and are monitored in forensic toxicology and anti-doping laboratories. Their abuse can affect heart rate, blood pressure, and fine motor control, prompting regulatory bodies such as WADA to include them on prohibited substance lists. Sensitive and reliable screening methods are therefore essential for both clinical and sports settings.

Study Objectives and Overview

This application note describes a comprehensive approach to detect and identify a panel of 16 common beta-blockers in human urine using ultra-performance liquid chromatography coupled with high-resolution, accurate-mass mass spectrometry (UPLC-MS). The goals were to achieve minimum required performance levels (MRPL) defined by WADA, to streamline sample preparation, and to automate data processing with UNIFI software.

Methodology and Instrumentation

Sample Preparation:

• A mixed methanolic standard of 16 beta-blockers (50 µg/mL) was prepared.
• Blank urine was spiked to final levels of 50, 100, 250, and 500 ng/mL.
• Samples were simply diluted five-fold with aqueous mobile phase prior to injection.

Chromatographic Conditions:

• System: ACQUITY UPLC I-Class with FTN autosampler.
• Column: ACQUITY UPLC HSS C18, 2.1 × 150 mm, 1.8 µm, at 50 °C.
• Mobile phase A: 5 mM ammonium formate, pH 3.0; B: acetonitrile with 0.1% formic acid.
• Gradient: 87% A to 50% A over 10 min, down to 5% A for 1.5 min, total run time 15 min; flow rate 0.4 mL/min; injection volume 10 µL.

Mass Spectrometry Conditions:

• Instrument: Xevo G2-S QTof with ESI+ source.
• Acquisition mode: MS^E with low (6 eV) and high collision energy (10–40 eV ramp).
• Mass range m/z 50–1000; scan time 0.1 s; reference mass leucine enkephalin.

Data Processing:

• UNIFI software with Forensic Toxicology Library (>1000 substances).
• Automated workflows classify findings as “Positive” (≤ 5 ppm mass error, ≥ 1 fragment, ±0.35 min RT) or “Tentative” (> 5 ppm ≤ 20 ppm).
• Isotopic pattern matching and apex-tracking algorithms enhance confidence.

Results and Discussion

All 16 beta-blockers were confidently identified at 50 ng/mL, meeting WADA MRPL. Chromatograms, accurate‐mass spectra, and fragment ion confirmation were presented via interactive plots and tables within UNIFI. Isotopic ratios (e.g., 32S/34S for sotalol) and mass accuracy metrics supported compound verification. Semi‐quantitative calibration curves (50–500 ng/mL) showed linear responses without internal standards, illustrating the system's dynamic range.

Benefits and Practical Applications

• High sensitivity and selectivity with minimal sample preparation.
• Automated data filtering reduces manual review time.
• Customizable reporting supports compliance with forensic and anti-doping guidelines.
• Comprehensive library matching enables broad screening capabilities.

Future Trends and Opportunities

Advances may include incorporation of isotopically labeled internal standards for fully quantitative assays, expansion of compound libraries to emerging designer beta-blockers, and integration with laboratory information management systems for streamlined workflows. Combining UPLC-MS^E with machine learning could further enhance identification accuracy and throughput.

Conclusion

The UPLC-MS^E workflow using UNIFI software provides a robust, automated solution for sensitive screening of beta-blockers in urine, fulfilling forensic and anti-doping requirements with high confidence and minimal analyst intervention.

References

1. Black JW, Crowther AF, Shanks RG, Smith LH, Dornhorst AC. A new adrenergic beta-receptor antagonist. Lancet. 1964;1(7342):1080–1081.
2. World Anti-Doping Agency. The World Anti-Doping Code: 2014 Prohibited List. 2014.
3. Rosano TG, Wood M, Ihenetu K, Swift TA. Drug screening in medical examiner casework by high resolution mass spectrometry (UPLC-MS^E-TOF). J Anal Toxicol. 2013;37(8):580–593.
4. Waters Corporation. Forensic Toxicology Screening. Brochure 720004830EN. 2014.